Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 November 2017 to 24 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Physical state/Appearance: Brown viscous liquid
Purity: >90%
Analytical monitoring:
yes
Details on sampling:
The molybdenum concentrations and stability of the test solutions were assessed by chemical analysis at 0 and 72 hours.
Vehicle:
no
Details on test solutions:
Due to the low aqueous solubility and the complex nature of the test item, the test medium was prepared as Water Accommodating Fraction (WAF) based on a the methods established in a preliminary mixing trial.

Nominal amounts of test item were each separately added to the surface of culture medium to give the 0.60, 1.9, 5.7, 17 and 50 mg/L loading rates WAF, respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer at a rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures were allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid depth siphoning (the first 75 to 100 mL discarded) to give the 0.60, 1.9, 5.7, 17 and 50 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium (Section 3.3). The master cultures were maintained in the laboratory under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 +/- 1 °C.

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 +/- 1 °C until the algal cell density was approximately 10^4 to 10^5 cells/mL.
Test type:
static
Water media type:
other: algal culture medium
Total exposure duration:
72 h
Test temperature:
24 +/- 1 °C
pH:
7.5 to 7.9
Nominal and measured concentrations:
Nominal concerntrations were 0 (control), 0.60, 1.9, 5.7, 17 and 50 mg/L loading rate WAF.

Analysis of samples collected from the 1.9 to 50 mg/L loading rate WAF solutions at 0 hours showed that measured concentrations ranged from 0.0024 mg/L to 0.17 mg/L as molybdenum, and at 72 hours showed that measured concentrations ranged from 0.0026 mg/L to 0.17 mg/L as molybdenum. At 0.60 mg/L loading rate WAF, analysis of the test preparations at 0 and 72 hours were below the Limit of Quantification (LOQ), determined to be 0.0020 mg/L as molybdenum.
Details on test conditions:
As in the range-finding tests 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.

The control group was maintained concurrently under identical conditions, but was not exposed to the test item.

Pre culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.23 x 10^6 cells per mL. Inoculation of 500 mL of test medium with 2.0 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs, incubated at 24 +/- 1 °C under continuous illumination provided by warm white lighting, and constantly shaken at approximately 150 rpm for 72 hours.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
11 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
growth rate
Remarks on result:
other: (95% confidence limits, 9.5 to 13 mg/L loading rate WAF)
Key result
Duration:
72 h
Dose descriptor:
NOELR
Remarks:
(and ErC10)
Effect conc.:
5.7 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate WAF
Basis for effect:
growth rate
Details on results:
There were no statistically significant differences in growth rate between the control and the 0.60, 1.9 and 5.7 mg/L loading rate WAF test groups (P >/= 0.05), however, growth rate at all other loading rates was significantly inhibited (P<0.05). The No Observed Effect Loading Rate (NOEL) based on growth rate was therefore 5.7 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOEL) based on growth rate was 17 mg/L loading rate WAF.

There were no statistically significant differences in yield between the control and 0.60 mg/L loading rate WAF (P >/= 0.05), however, yield in all other loading rates was significantly lower (P<0.05). The NOEL based on yield was therefore 0.60 mg/L loading rate WAF and the LOEL based on yield was 1.9 mg/L loading rate WAF.

There were no statistically significant differences in biomass between the control and 0.60 mg/L loading rate WAF (P >/= 0.05), however, biomass in all other loading rates was significantly less (P<0.05). The NOEL based on biomass was 0.60 mg/L loading rate WAF and the LOEL based on biomass was 1.9 mg/L loading rate WAF.

The cell concentration of the control cultures increased by a factor of 319 after 72 hours. The mean coefficient of variation for section by section specific growth rate for the control cultures was 16% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%. The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 4% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

At the start of the test all control and test cultures were observed to be clear colorless solutions. After 72 hours, all control, 0.60, 1.9 and 5.7 mg/L loading rate WAF test cultures were observed to be green dispersion and no abnormalities were detected. The 17 and 50 mg/L loading rate WAF test cultures were observed to be clear colorless solutions after the 72-Hour test period.

Results with reference substance (positive control):
A positive control (performed in-house), using potassium dichromate as the reference item, was conducted on November 6 - 9, 2017, at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.  Exposure conditions and data evaluation for the positive control were similar to those in the definitive test. Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:

ErC50 (0 to 72 hour): 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour): 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L

NOErC (based on growth rate): 0.25 mg/L
NOEyC (based on yield): 0.25 mg/L

LOErC (based on growth rate): 0.50 mg/L
LOEyC (based on yield): 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
Statistical analyses of the growth rate, yield and biomass data were carried out for the control and all loading rates using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955).

Inhibition of Growth Rate, Yield and Biomass in the Definitive Test Nominal Loading Rate

 

 

Growth Rate (cells/mL/hour)

Yield (cells/mL)

Biomass Integral

Nominal Loading Rate
(mg/L)

Rep

0 to 72 Hour

%
Inhibition

0 to 72 Hour

% Inhibition

0 to72 Hour

%
Inhibition

Control

R1

0.084

-

2.04E+06

-

3.49E+07

-

.

R2

0.080

 

1.60E+06

 

2.72E+07

 

.

R3

0.077

 

1.23E+06

 

2.19E+07

 

.

R4

0.080

 

1.53E+06

 

2.63E+07

 

.

R5

0.082

 

1.79E+06

 

3.05E+07

 

.

R6

0.078

 

1.35E+06

 

2.37E+07

 

.

Mean

0.080

 

1.59E+06

 

2.74E+07

 

.

SD

0.003

 

2.96E+05

 

4.69E+06

 

0.60

R1

0.081

[1]

1.68E+06

 

2.83E+07

[3]

.

R2

0.079

1

1.51E+06

 

2.57E+07

6

.

R3

0.077

4

1.23E+06

 

2.12E+07

23

.

Mean

0.079

1

1.47E+06

7

2.51E+07

9

.

SD

0.002

 

2.28E+05

 

3.57E+06

 

1.9

R1

0.077

4

1.26E+06

 

2.16E+07

21

.

R2

0.076

5

1.21E+06

 

2.09E+07

24

.

R3

0.072

10

9.02E+05

 

1.63E+07

40

.

Mean

0.075

6

1.13E+06

29

1.96E+07

28

.

SD

0.003

 

1.95E+05

 

2.87E+06

 

5.7

R1

0.073

9

9.21E+05

 

1.66E+07

39

.

R2

0.072

10

9.13E+05

 

1.62E+07

41

.

R3

0.070

13

7.71E+05

 

1.40E+07

49

.

Mean

0.072

11

8.68E+05

45

1.56E+07

43

.

SD

0.002

 

8.42E+04

 

1.43E+06

 

17

R1

0.028

65

3.35E+04

 

4.77E+05

98

.

R2

0.013

84

7.38E+03

 

2.52E+03

100

.

R3

0.008

90

3.95E+03

 

-8.32E+04

100

.

Mean

0.016

80

1.50E+04

99

1.32E+05

99

.

SD

0.010

 

1.62E+04

 

3.02E+05

 

50

R1

0.016

80

1.13E+04

 

2.70E+05

99

.

R2

0.007

91

3.15E+03

 

-8.32E+04

100

.

R3

0.008

90

3.98E+03

 

-1.52E+04

100

.

Mean

0.010

87

6.16E+03

100

5.71E+04

100

.

SD

0.005

 

4.51E+03

 

1.87E+05

 

*In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R = Replicate

- = Not applicable

SD = Standard deviation

[ ] = Increase in growth compared to controls

Growth Effects on Pseudokirchneriella subcapitata following 72‑Hour Exposure

Response Variable

EL50
(mg/L Loading Rate WAF)

95% Confidence Limits
(mg/L Loading Rate WAF)

NOEL
(mg/L)

LOEL
(mg/L)

Growth Rate

11

9.4

to

13

5.7

17

Yield

4.7

3.8

to

5.9

0.6

1.9

Biomass

4.9

3.6

to

6.7

0.6

1.9

Validity criteria fulfilled:
yes
Conclusions:
Exposure of Pseudokirchneriella subcapitata to the test item for 72 hours resulted in an EL50 of 11 mg/L Loading Rate WAF, a NOEL of 5.7 mg/L Loading Rate WAF, and a LOEL of 17 mg/L Loading Rate WAF, based on growth rate.
Executive summary:

A study was conducted according to OECD Guideline 201 and EC Method C.3, under GLP conditions, to assess the effects of the test item on the growth of the freshwater green alga, Pseudokirchneriella subcapitata.  

 

Due to the low aqueous solubility and complex nature of the test item, the test solutions were prepared as Water Accommodated Fractions (WAF).

 

Following preliminary range finding tests, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item at nominal loading rates of 0.60, 1.9, 5.7, 17 and 50 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 ˚C. Samples of the test solutions were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

 

At 1.9 to 50 mg/L loading rate WAF, analysis of the test solutions at 0 hours showed that measured concentrations of between 0.0024 and 0.17 mg/L as molybdenum were obtained. Measured concentrations were similar at 72 hours, ranging from 0.0026 mg/L to 0.17 mg/L as molybdenum were obtained. At 0.60 mg/L loading rate WAF, analysis of the test preparations at 0 and 72 hours were below the Limit of Quantification (LOQ), determined to be 0.0020 mg/L as molybdenum. The dissolved test item may have been comprised of one or several components of the test item. Given that the toxicity cannot be attributed to a single component or a mixture of components, the results were based on nominal loading rates only.

 

Exposure of Pseudokirchneriella subcapitata to the test item for 72 hours resulted in an ErL50 of 11 mg/L Loading Rate WAF, a NOErL (and ErC10) of 5.7 mg/L Loading Rate WAF, and a LOEL of 17 mg/L Loading Rate WAF, based on growth rate.

Description of key information

A study was conducted according to OECD Guideline 201 and EC Method C.3, under GLP conditions, to assess the effects of the test item on the growth of the freshwater green alga, Pseudokirchneriella subcapitata. Exposure of Pseudokirchneriella subcapitata to the test item for 72 hours resulted in an EL50 of 11 mg/L loading rate WAF, an EC10 (and NOEL) of 5.7 mg/L loading rate WAF, and a LOEL of 17 mg/L loading rate WAF, based on growth rate.

Key value for chemical safety assessment

EC50 for freshwater algae:
11 mg/L
EC10 or NOEC for freshwater algae:
5.2 mg/L

Additional information